Mycoplasma pneumoniae: A prokaryote which consumes oxygen and generates superoxide but which lacks superoxide dismutase

Superoxide dismutase and catalase were not detected in $\text{M}$. $\text{pneumoniae}$ and several other species of $\text{Mycoplasma}$ some of which consume oxygen and secrete H₂O₂. $\text{M}$. $\text{pneumoniae}$ in suspension formed O₂^? in the presence of NADH and flavins and extracts of $\text{M}$. $\text{pneumoniae}$ formed O₂^? in the presence of either NADH or NADPH. The lack of superoxide dismutase in $\text{M}$. $\text{pneumoniae}$ could not be attributed to superoxide dismutase in the complex medium in which the organisms were grown because organisms grown in medium in which the superoxide dismutase had been inactivated by heat still contained undetectable amounts. Mycoplasmas appear to be an exception to the rule that organisms which consume O₂ synthesize superoxide dismutase.     

Kinetics of subunit dissociation of partially oxygenated hemoglobin determined by haptoglobin binding

Human haptoglobin 1-1 binds very rapidly to hemoglobin dimers but not to tetramers. We have studied the binding kinetics of partially oxygenated Hb A to haptoglobin 1-1. Under the oxygenation conditions used for the measurement of the K₁ of oxygenation ($\text{Hb O}{}_2\text{Hb}\text{≤ 1\%}$, pO₂ ≤ 0.5 mm Hg), the dissociation kinetics were found to be 50 times faster than that of deoxy Hb A. This result suggested that the binding of one molecule of oxygen to hemoglobin tetramer changed the quaternary structure of the intersubunit α₁β₂ contact surface.     

The relative effectiveness of .OH,H2O2,O2−, and reducing free radicals in causing damage to biomembranes: A study of radiation damage to erythrocyte ghosts using selective free radical scavengers

The relative effectiveness of oxidizing (^.OH, H₂O₂), ambivalent (O₂^?) and reducing free radicals (e^? and CO₂^?) in causing damage to membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membranebound glyceraldehyde-3-phosphate dehydrogenase ($\text{R}$(enz)) were measured and the rates of damage to membranes ($\text{R}$(mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. ^.OH, O₂^? and H₂ O₂ were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e^? and CO₂^? were the least effective. $\text{R}$(enz) values of O₂^? and H₂O₂ were 10-times and of ^.OH 15-times that of e^?. $\text{R}$(mb) values were quite similar for e^? and H₂O₂ (about twice that of O₂^?), while that of ^.OH was 3-times that of O₂^?. Hence, with respect to $\text{R}$(mb): ${}^{\mathrm{.}}\text{OH}\text{>}\text{e}{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{O}{}_2{}^{\mathrm{?}}$ , and with respect to $\text{R}$(enz): ${}^{\mathrm{.}}\text{OH}\text{>}\text{O}{}_2{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{e}{}^{\mathrm{?}}$. The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H₂O₂ added as a chemical reagent and H₂O₂ formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H₂O₂ but markedly susceptible to the latter.     

Preparation of O2-evolving Photosystem-II subchloroplasts from spinach

An O₂-evolving Photosystem II subchloroplast preparation was obtained from spinach chloroplasts, using low concentrations of digitonin and Triton X-100. The preparation showed an O₂ evolution activity equivalent to 20% of the uncoupled rate of fresh broken chloroplasts, but had no significant Photosystem-I-dependent O₂ uptake activity. The preparation showed a chlorophyll $\text{a}\text{b}$ ratio of 1.9 and a $\text{P-700}\text{chlorophyll}$ ratio of $\text{1}\text{2400}$. Absorption spectra at room temperature and fluorescence emission spectra of chlorophyll at 77 K suggested a significant decrease in Photosystem I antenna chlorophylls in the O₂-evolving Photosystem II preparation.     

Do copper ions influence the reduction of ferricytochrome C by O−2?

Recently, it was suggested that the measured rate of reduction of ferricyto chrome $\text{C}$ by O^?₂ below pH 8, was too high in the presence of high concentrations of formate (Koppenol, W.H., Van Buuren, K.J.H., Butler J. and Braams, R. (1976) Biochim. Biophys. Acta 449, 157–168).The high values were attributed to the presence of impurities of copper, which compete for O^?₂. This assumption is consistent with either a decrease in the reduction yield of ferricytochrome $\text{C}$ in the presence of copper, or with a very fast reaction of Cu(I) with ferricytochrome $\text{C}$.It was previously shown by us and by others that the reduction yield of ferricytochrome $\text{C}$ by O^?₂ is 100%. We measured the rate of reduction of ferricytochrome $\text{C}$ by Cu(I), and found that this reaction is slow: $\text{k = (1.5±0.5) · 10}{}^3\text{M}{}^{\mathrm{?1}}\text{) ·}\text{s}{}^{\mathrm{?1}}$.Therefore, our results rule out the possibility that below pH 8 copper impurities affect the measured rate constant of the reduction of ferricytochrome $\text{C}$ by O^?₂.     

Kinetic studies of 18O exchange from inorganic phosphate catalyzed by Mg2+-activated unadenylylated glutamine synthetase (E. coli w).

Oxygen-18 exchange out of [¹⁸O]Pi catalyzed by Mg²⁺-activated unadenylated glutamine synthetase from $\text{E.}$$\text{coli}$ was followed by ³¹P-NMR in the presence of the other substrates, ADP and L-glutamine. The pattern of the ${}^{16}\text{O}{}^{18}\text{O}$ in the species P¹⁸O₄, P¹⁸O₃¹⁶O₁, P¹⁸O₂¹⁶O₂, P¹⁸O₁¹⁶O₃, P¹⁶O₄ during the exchange followed a binomial distribution consistent with indiscriminate removal of any of the four oxygens of P_i. The rate constant for ${}^{16}\text{O}{}^{18}\text{O}$ exchange was 410±40 min^?1 while the rate constant for net reaction (ATP formation) was 62±4 min^?1. Thus exchange proceeds ～7 times faster than net reaction, a finding in accord with that of Stokes and Boyer ($\text{J.}$$\text{Biol.}$$\text{Chem.}$ (1976) $\text{251}$, 5558) for the Mn²⁺-activated adenylylated glutamine synthetase. A model for the overall catalytic events first derived from rapid kinetic fluorescence experiments (Rhee and Chock, Proc. Natl. Acad. Sci. USA, (1976) $\text{73}$, 476) was successfully used to fit the oxygen exchange data in this paper.     

Early identification of non-pregnant and pregnant ewes in the field using circulating progesterone concentration

A method for early indentification of non-pregnant and pregnant ewes is described. It is applicable to field research situations where mating data for individual ewes cannot be collected and requires three plasma progesterone measurements from each ewe over a 12-days period.Ewes were diagnosed non-pregnant according to whether their lowest progesterone concentration (p) was below or above a “discriminatory value”. This value was chosen after examining the overall frequency distribution of values of log₁₀p.In experiment 1, $\text{25}\text{27}$ non-pregnant ewes and $\text{64}\text{65}$ pregnant ewes (20 to 31 days post-mating) were diagnosed correctly (96.7% accuracy). In experiment 2, $\text{40}\text{41}$ non-pregnant ews and $\text{45}\text{48}$ pregnant ewes (21 to 34 days post-mating) were diagnosed correctly (95.5% accuracy).     

Iron(II)-bleomycin. Biochemical and spectral properties in the presence of radical scavengers

Consistent with a recent literature report (Repine, J. E. $\text{et}$$\text{al.}$ (1981) $\text{Proc.}$$\text{Nat.}$$\text{Acad.}$$\text{Sci.}$$\text{USA}$$\text{7}\text{?}$$\text{8}\text{?}$, 1001–1003), the release of [³H]-thymine from PM-2 DNA by Fe(II)-H₂O₂-generated ·OH was suppressed by dimethyl sulfoxide. In contrast, DMSO did not affect [³H]-thymine release mediated by Fe(II)-bleomycin. Under aerobic conditions in the presence of $\text{t}$-butyl phenylnitrone, Fe(II)-BLM produces an epr signal that has been presumed to arise by transfer of ·OH or $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ from the “active complex” of bleomycin to the spin trap. Remarkably, high concentrations (80 mM) of PBN had no effect on the ability of Fe(II)-BLM to solubilize [³H]-thymine, although the ability of authentic ·OH to degrade DNA was completely suppressed under these condition. The suproxide dismutase catalyst tetrakis(4-N-methylpyridyl)porphineiron(III) also failed to suppress BLM-mediated DNA degradation. Moreover, the epr signal observed with 1.6 mM Fe(II)-BLM in the presence of 80 mM PBN was found to be much less intense than that produced by 1.6 mM Fe(II) and 290 mM H₂O₂, but equivalent in intensity to that obtained with 45 mM Fe(II) and exoess H₂O₂. We conclude that the fragmentation of DNA produced by Fe(II)-BLM can be due neither to free ·OH nor to $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$. We suggest that DNA degradation is initiated by an “active complex” consisting of BLM, metal and oxygen that functions by abstracting H· from susceptible sites on DNA.     

Adaptation of Nodulated Soybean (Glycine max L. Merr.) to Growth in Rhizospheres Containing Nonambient pO(2)

Nodulated soybean (Glycine max L. Merr. cv White Eye inoculated with Bradyrhizobium japonicum strain CB 1809) plants were cultured in the absence of combined N from 8 to 28 days with their root systems maintained continuously in 1, 2.5, 5, 10, 20, 40, 60, or 80% O₂ (volume/volume) in N₂. Plant dry matter yield was unaffected by partial pressure of oxygen (pO₂) and N₂ fixation showed a broad plateau of maximum activity from 2.5 to 40 or 60% O₂. Slight inhibition of nitrogenase activity occurred at 1% O₂ and as much as 50% inhibition occurred at 80% O₂. Low pO₂ (less than 10%) decreased nodule mass on plants, but this was compensated for by those nodules having higher specific nitrogenase activities. Synthesis and export of ureides in xylem was maintained at a high level (70-95% of total soluble N in exudate) over the range of pO₂ used. Measurements of nitrogenase (EC 1.7.99.2) activity by acetylene reduction indicated that adaptation of nodules to low pO₂ was largely due to changes in ventilation characteristics and involved increased permeability to gases in those grown in subambient pO₂ and decreased permeability in those from plants cultured with their roots in pO₂ greater than ambient. A range of structural alterations in nodules resulting from low pO₂ were identified. These included increased frequency of lenticels, decreased nodule size, increased volume of cortex relative to the infected central tissue of the nodule, as well as changes in the size and frequency of extracellular voids in all tissues. In nodules grown in air, the inner cortex differentiated a layer of four or five cells which formed a band, 40 to 50 micrometers thick, lacking extracellular voids. This was reduced in nodules grown in low pO₂ comprising one or two cell layers and being 10 to 20 micrometers thick in those from 1% O₂. Long-term adaptation to different external pO₂ involved changes which modify diffusive resistance and are additional to adjustments in the variable diffusion barrier.     

Superoxide-dependent formation of hydroxyl radicals: Detection of hydroxyl radicals by the hydroxylation of aromatic compounds

A mixture of xanthine or hypoxanthine and xanthine oxidase generates the superoxide radical, O₂^$\text{?}$, and H₂O₂. In the presence of iron salts, O₂^$\text{?}$ and H₂O₂ can interact to produce the hydroxyl radical, OH·. Superoxide-dependent formation of OH· can be measured by its ability to hydroxylate salicylate as followed by an improved colorimetric assay described in this paper. A more accurate analysis of OH· can be obtained using its ability to hydroxylate phenol, the hydroxylated products being separated and measured after derivatization using gas-liquid chromatography and electron-capture detection. The derivatization and separation techniques are described.     

Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system.

Ascorbic acid was found to be oxidized by O₂^$\text{?}$ which was generated by the xanthine-xanthine oxidase system. From a kinetic analysis of the inhibition of this reaction by superoxide dismutase, the second-order rate constant for the reaction between ascorbic acid and O₂^? at pH 7.4 was estimated to be 2.7 × 10⁵ M^?1 sec^?1. A function of ascorbic acid as a defense against O₂^? is presented.     

The duplication of arginine catabolism and the meaning of the two ornithine carbamoyltransferases in Bacillus licheniformis.

Arginine deiminase and carbamate kinase activities are shown to be present in cell-free extracts of $\text{Bacillus licheniformis}$. This gives the rationale for the occurrence of an arginine-inducible ornithine carbamoyltransferase in this organism and suggests that, $\text{in vivo}$ and under the proper conditions, this enzyme is able to catalyze the phosphorolysis of citrulline. This also shows that this Bacillus species has two arginine catabolic pathways. This duplication appears to be under the control of O₂.     

Superoxide reduction as a mechanism of ascorbate-stimulated oxygen uptake by isolated chloroplasts

Addition of 1m$\text{M}$ ascorbate to isolated chloroplasts with methyl viologen (MV) as electron acceptor trebled the rate of oxygen uptake and decreased the $\text{ADP}\text{O}$ ratio to a third of that with no ascorbate present. These effects of ascorbate were reversed by superoxide dismutase (SOD), which in the absence of ascorbate had little effect on O₂ uptake or $\text{ADP}\text{O}$ ratio. A chloroplast-associated SOD activity equivalent to 500 units/mg chlorophyll was detected. The effects of ascorbate and SOD on O₂ uptake were similar in both coupled and uncoupled chloroplasts. The results are consistent with the hypothesis that ascorbate stimulates O₂ uptake by reduction of superoxide, which is formed by autoxidation of the added electron acceptor (MV), and which dismutates in the absence of ascorbate. Ascorbate does not seem to stimulate O₂ uptake by replacing water as the photosystem II donor.     

Calorimetric measurement of stepwise enthalpy changes for the binding of four oxygens to adult human hemoglobin

The successive enthalpy changes for the four steps of oxygen binding by diphosphoglycerate-free adult human hemoglobin have been measured by direct calorimetry at pH 7.4 and 6°. Average results in kcal/(mole O₂) are: ΔH₁ = ?25.1 ± 2.8; ΔH₂ = ?12.6 ± 3.0, ΔH₃ = ?12.5 ± 3.0, and ΔH₄ = ?10.1 ± 1.4. These results imply a substantial temperature dependence for the cooperativity of O₂ binding by the protein and generally resemble the van't Hoff results by Roughton $\text{et al}$. [Roy. Soc. of London Proc., $\text{B 144}$, 29 (1955)] for sheep hemoglobin at pH 9.1 and a temperature range of 2° to 19°.     

Dual action of ionophore A23187 on intact chloroplasts

1. Ionophore A23187 induces uncoupling of potassium ferricyanide-dependent O₂ evolution by envelope-free chloroplasts and oxaloacetate-dependent O₂ evolution by intact chloroplasts. The half maximal concentration ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for stimulation of oxygen evolution in both cases is approximately 4 μM · 100 μg chlorophyll · ml^?1.2. Ionophore A23187 also induces inhibition of CO₂ and 3-phosphoglycerate-dependent O₂ evolution by intact chloroplasts in the presence of 3 mM MgCl₂. The half maximal concentrations ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for inhibition of O₂ evolution are 3 μM and 5 μM respectively · 100 μg^?1 chlorophyll · ml^?1.3. A very high concentration of ionophore A23187 (10 μM · 20 μg^?1 chlorophyll · ml^?1) plus 0.1 mM EDTA lowers the fluorescence yield of intact chloroplasts suspended in a cation-free medium in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating loss of divalent cation from the diffuse double layers of the thylakoid membranes.4. These results are discussed in relation to ionophore A23187-induced divalent cation/proton exchange at both the thylakoid and the envelope membranes of intact chloroplasts.     

Complete stoichiometry of free NADPH oxidation in liver microsomes

The stoichiometry of free NADPH oxidation in phenobarbital induced rabbit liver microsomes was measured by means of registering the rates of NADPH, H⁺ and O₂ consumption and $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and H₂O₂ production. $\text{ΔO}{}_2{}^{\mathrm{<mtext>?</mtext>}}\text{:ΔH}{}_2\text{O}{}_2$ ratio is approximately I indicating that about half H₂O₂ results from $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ dismutation, the second half being formed directly. ΔNADPH:ΔH₂O₂ and ΔO₂:ΔH₂O₂ ratios exceed I and therefore another product of the reaction is water. The fact that the ratio (ΔNADPH-ΔH₂O₂):(ΔO₂-ΔH₂O₂) is 2 allows one to consider direct 4-electron O₂ reduction as the major way of water formation rather than endogenous substrate hydroxylation.     

Dibutyryl cGMP: inhibitor of the effect of cholecystokinin and gastrin on the guinea pig gallbladder in vitro

N², O²-di-butyryl guanosine 3′:5′ monophosphate (Bt₂ cGMP), a known competitive and selective inhibitor of the effect of cholecystokinin on the pancreatic acinar cells $\text{in}$$\text{vitro}$ was tested for its effect on the guinea pig gallbladder $\text{in}$$\text{vitro}$. Bt₂ cGMP inhibited competitively the contractile effect of cholecystokinin octapeptide, and also inhibited the contraction induced by sulfated gastrin-17. Bt₂ cGMP failed to inhibit the contraction induced by bombesin, acetylcholine or histamine. The 8-bromo derivative of cGMP and the dibutyryl derivative of cAMP did not affect contraction stimulated by cholecystokinin octapeptide. Since it is specific for gastrincholecystokinin peptides, and not restricted to the pancreas, Bt₂ cGMP could be used to recognize the action of these peptides.     

Photobiochemistry in the dark: photohemolysis of red cells sensitized by chlorpromazine-bioenergized triplet acetone system

Chlorpromazine is decomposed when it is treated with bioenergized triplet acetone from the 2-methylpropanal/red cells/O₂ system, forming chlorpromazine-5-oxide, with a concomitant strong hemolytic effect observed by a spectrophotometric method. Experiments with external superoxide dismutase, catalase, benzoate and bicarbonate indicate the absence of $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$, H₂O₂ and OH· species as the precursor of the hemolytic effect.Comparison between the 2-methylpropanal/peroxidase/O₂ system and the 2-methylpropanal/red cells/O₂ system in the presence of chlorpromazine, indicate that essentially the same type of mechanism occurs in both cases.These results could explain the $\text{in}$$\text{vivo}$ hemolytic and toxic effect of chlorpromazine in the dark.     

The ATP/2e ratio during photosynthesis in intact spinach chloroplasts.

Addition of ribose-5-phosphate to intact spinach chloroplasts in the absence of added P_i resulted in a conversion of part of the Benson-Calvin cycle into a linear sequence so that triose phosphate accumulated during CO₂ fixation stoichiometrically with the O₂ evolved (triose phosphate / O₂ ratio was 2.0). The fortunate consequence of this effect is that the $\text{ATP}\text{2e}$ ratio may be calculated from the 3-phosphoglycerate and triose phosphate accumulated and the O₂ evolved. In this way the $\text{ATP}\text{2e}$ ratio was shown to be 2.0, with cyclic or pseudocyclic phosphorylation contributing less than 9% to the total phosphorylation.     

Effects of 19-hydroxy-prostaglandins on oviductal and uterine motility

The effects of 19-hydroxy-prostaglandins (19-OH-PGs) were tested $\text{in}$$\text{vivo}$ on the rabbit oviduct and uterus and on the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) uterus. The 19-OH-PGEs suppressed spontaneous oviductal and uterine activity in the rabbit. The qualitative effect on the rabbit oviduct of 19-OH-PGEs was similar to that of PGE₂. However, the typical response of the rabbit uterus to PGE₂ was an increase in muscle activity. With regard to the rabbit oviduct, 19(R)-OH-PGE₂ was as potent as PGE₂, but 19(S)-OH-PGE₂ was approximately $\text{1}\text{2}$ as potent as PGE₂. Based on the dose of 19-OH-PGEs usually required to cause a minimal suppression and the dose of PGE₂ required to cause a minimal stimulation of rabbit uterine activity, 19(R)-OH-PGE₂ was twice as potent as PGE₂ while 19(S)-OH-PGE₂ was $\text{1}\text{2}$ as potent as PGE₂. Stimulatory effects on the rabbit oviduct and uterus were observed following administration of 19-OH-PGEs and PGF_2α. The potency on the rabbit oviduct of 19(S)-OH-PGF_2α was about $\text{1}\text{5}$ to $\text{1}\text{10}$ that of PGF_2α; the potency of 19(R)-OH-PGF_2α was about $\text{1}\text{10}$ to $\text{1}\text{20}$ that of PGF_2α. Both 19-OH-PGFs were approximately $\text{1}\text{5}$ to $\text{1}\text{10}$ as potent as PGF_2α on the rabbit uterus. At the doses tested 19-OH-PGFs were inactive on the monkey uterus. Thus, these compounds are at least $\text{1}\text{5}$ as active as PGF_2α. In contrast, 19(R)-OH-PGE₂ had approximately the same potency as PGE₂ in stimulating monkey uterine activity; but 19(S)-OH-PGE₂ was approximately $\text{1}\text{3}$ as potent as PGE₂.